Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells

<div><h3>Background</h3><p>Epidemiological case-control studies have revealed associations between mitochondrial haplogroups and the onset and/or progression of various multifactorial diseases. For instance, mitochondrial haplogroup T was previously shown to be associated with vascular diseases, including coronary artery disease and diabetic retinopathy. In contrast, haplogroup H, the most frequent haplogroup in Europe, is often found to be more prevalent in healthy control subjects than in patient study groups. However, justifications for the assumption that haplogroups are functionally distinct are rare. Therefore, we attempted to compare differences in mitochondrial function between haplogroup H and T cybrids.</p> <h3>Methodology/Principal Findings</h3><p>Mitochondrial haplogroup H and T cybrids were generated by fusion of HEK293 cells devoid of mitochondrial DNA with isolated thrombocytes of individuals with the respective haplogroups. These cybrid cells were analyzed for oxidative phosphorylation (OXPHOS) enzyme activities, mitochondrial DNA (mtDNA) copy number, growth rate and susceptibility to reactive oxygen species (ROS). We observed that haplogroup T cybrids have higher survival rate when challenged with hydrogen peroxide, indicating a higher capability to cope with oxidative stress.</p> <h3>Conclusions/Significance</h3><p>The results of this study show that functional differences exist between HEK293 cybrid cells which differ in mitochondrial genomic background.</p> </div

Growth curves of mitochondrial haplogroup-specific cybrid cells.

<p>The number of cells on the days given were normalized to the number of cells on day two and determined as growth rate. (A) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in glucose medium. (B) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in galactose medium. Mean values of growth rates are given; error bars: standard deviation; *p<0.05.</p

Enzymatic activities of citrate synthase and oxidative phosphorylation complexes I – V in haplogroup H and T cybrid cells.

a<p>Values are given as mean ± standard deviation (SD).</p>b<p>P-value: Independent samples t-test.</p>c<p>Reported previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone-0052367-g002" target="_blank">Figure 2</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-Mueller2" target="_blank">[31]</a>.</p><p>Enzymatic activity measurements were made on isolated mitochondria of cells grown in glucose medium with antibiotics, and on cells with five to 15 passages after the cybridization process.</p

Cell survival after treatment with H₂O₂.

<p>Cell survival was measured 24 hours after H₂O₂ treatment and calculated as a percentage of the ratio between treated and untreated cells (% cell survival). (A) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 250 µM to 475 µM H₂O₂ in glucose medium without serum and without sodium pyruvate. (B) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 100 µM to 250 µM H₂O₂ in galactose medium without serum and without sodium pyruvate. Mean values of % cell survival are given; error bars: standard deviation; *p<0.05.</p

Results of TaqMan qPCR analysis of HEK H and HEK T cybrid competitive co-cultures.

<p>After 10, 20 and 30 days (d10, d20, d30) of co-culture, isolated DNA of the cell mixtures was analyzed using TaqMan qPCR. ΔC_t values were calculated by subtraction of the mean C_t value of the FAM signal (probe recognizing haplogroup T) from the mean C_t value of the VIC signal (probe recognizing haplogroup H). ΔΔC_t values were calculated by subtraction of the mean ΔC_t values of the original cell mixtures (n = 36; day zero) from the mean ΔC_t values of all co-cultures at days 10, 20 or 30 (n = 36; except for d30 in galactose: n = 35). Dominance of haplogroup H results in a negative ΔΔC_t value and is presented as gray bars, whereas dominance of haplogroup T results in a positive ΔΔC_t value and is presented as black bars. (A) ΔΔC_t values of competitive co-cultures cultivated in glucose medium, at days 10, 20 and 30. (B) ΔΔC_t values of competitive co-cultures cultivated in galactose medium, at days 10, 20 and 30. Mean ΔΔC_t values are given; error bars: standard deviation.</p

Figure 1. Phylogenetic tree of haplogroup H and T subsets.

<p>The phylogenetic tree was constructed according to phylotree.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-vanOven1" target="_blank">[32]</a>. ^aC16296T did not appear in the mtDNA sequence of cybrid T1. ^bBases of the Revised Cambridge Reference Sequence that appear in HEK T cybrids as polymorphisms diagnostic for non-H haplogroups (m.73A>G, m.2706A>G, m.7028C>T, m.11719G>A and m.14766C>T).</p

Mitochondrial DNA copy number in cybrid cells.

<p>(A) Comparison of all cybrid clones cultivated in glucose medium and galactose medium. (B) Comparison of HEK H and HEK T cybrids cultivated in glucose medium. (C) Comparison of HEK H and HEK T cybrids cultivated in galactose medium. Mean values of copy numbers are given; error bars: standard deviation; *p<0.05.</p

Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

<div><p>Introduction</p><p>Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.</p><p>Methods</p><p>Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).</p><p>Results</p><p>Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.</p><p>Conclusions</p><p>Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.</p></div

Immunohistochemical (IHC) staining of mitochondrial OXPHOS complexes I-V and VDAC in SH-SY5Y xenograft tumors.

<p>IHC staining of NB sections were scored on a scale from 0–3 as described in the methods section. The CR-KD group showed a significant decrease in complex I staining. All other evaluated parameters were unaffected by dietary changes. Voltage-dependent ion channel (VDAC) protein levels are used as a surrogate marker of mitochondrial mass. Statistics: ANOVA (p <0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD group.</p

Ketogenic diet and calorie restriction reduce tumor growth and prolong survival in a NB xenograft model.

<p>After establishing tumors on the right flank of CD-1nu mice, the mice were randomized to diet groups as indicated. Tumor volume was measured twice weekly. A) For SH-SY5Y xenografts at day 19, the tumors of all diet groups showed significant growth inhibition compared to the SD group (CR-SD p = 0.001, KD p<0.001, CR-KD p<0.001). B) At day 33, SK-N-BE(2) tumor growth was significantly inhibited by CR (CR-SD p = 0.040, CR-KD p = 0.004). Inhibition of tumor growth was less pronounced in the KD group (p = 0.918). C) SH-SY5Y and D) SK-N-BE(2) show the results of Kaplan-Meier survival analysis of the corresponding treatment groups. Survival of mice with SH-SY5Y tumors at day 22 on SD was 0% compared to 75% on CR-SD (p<0.001), 50% on KD (p<0.001) and 100% on CR-KD (p<0.001). Survival of mice with SK-N-BE(2) xenografts at day 33 on SD was 36% compared to 83% on CR-SD (p = 0.017), 73% on KD (p = 0.09) and 100% on CR–KD (p<0.001). A, B) Data points for tumor growth curves represent mean values ± SEM of the corresponding diet group (n = 8–11). Statistics: ANOVA (p<0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. C, D) Survival is expressed by the Kaplan–Meier method and differences between groups were determined in a univariate analysis with the log-rank test. Death is coded: tumor volume above 3000 mm³, tumor ulceration or impaired health condition. Diet groups are compared to the corresponding SD. * p≤0.05; ** p≤0.01; *** p≤0.001.</p